Design, Synthesis, and Biological Evaluation of Sulfonamide Methoxypyridine Derivatives as Novel PI3K/mTOR Dual Inhibitors

Phosphatidylinositol 3-kinase (PI3K) plays an important role in cell proliferation, survival, migration, and metabolism, and has become an effective target for cancer treatment. Meanwhile, inhibiting both PI3K and mammalian rapamycin receptor (mTOR) can simultaneously improve the efficiency of anti-tumor therapy. Herein, a series of 36 sulfonamide methoxypyridine derivatives with three different aromatic skeletons were synthesized as novel potent PI3K/mTOR dual inhibitors based on a scaffold hopping strategy. Enzyme inhibition assay and cell anti-proliferation assay were employed to assess all derivatives. Then, the effects of the most potent inhibitor on cell cycle and apoptosis were performed. Furthermore, the phosphorylation level of AKT, an important downstream effector of PI3K, was evaluated by Western blot assay. Finally, molecular docking was used to confirm the binding mode with PI3Kα and mTOR. Among them, 22c with the quinoline core showed strong PI3Kα kinase inhibitory activity (IC50 = 0.22 nM) and mTOR kinase inhibitory activity (IC50 = 23 nM). 22c also showed a strong proliferation inhibitory activity, both in MCF-7 cells (IC50 = 130 nM) and HCT-116 cells (IC50 = 20 nM). 22c could effectively cause cell cycle arrest in G0/G1 phase and induce apoptosis of HCT-116 cells. Western blot assay showed that 22c could decrease the phosphorylation of AKT at a low concentration. The results of the modeling docking study also confirmed the binding mode of 22c with PI3Kα and mTOR. Hence, 22c is a promising PI3K/mTOR dual inhibitor, which is worthy of further research in the area.


Optimization Strategy
Based on the co-crystal structures of the lipid kinase PI3Kα and structures of some known PI3K/mTOR dual inhibitors, it could be found that the structures mainly consisted of three parts (as shown in Figure 2): part A for the affinity binding pocket, part B for the hinge binding pocket, and part C for the ribose binding pocket. Parts A and B were essential to the activity, and the optimization of part C could improve the metabolic stability and oral bioavailability. The available results suggested [14] that when the structure of part A was 2,4-difluoro-N-(2-methoxypyridin-3-yl) benzenesulfonamide, it had the strongest PI3K inhibitory activity. Therefore, the optimization was mainly in parts Kim et al. [15] reported HS-173, a new PI3K inhibitor of imidazo [1,2-a]pyridine, which showed excellent enzyme inhibition at about 0.8 nM. Fan et al. [16] synthesized compound 1 with an amide moiety to significantly improve the metabolic stability while retaining the inhibitory activity. Yu et al. [17] further improved the design and reported compound 2 containing a hetero-linker in the structure with an increased activity of up to an IC 50 value of 0.2 nM for PI3K, and it showed inhibition in HCT-116 cells at 0.01 µM.
Pharmaceuticals 2023, 16, 461 3 of 22 In this paper, we designed and synthesized three series of compounds as potent PI3K/mTOR dual inhibitors containing fragments of benzo [4,5]thiopheno [3,2-d]pyrimidine, pyridine [2,3-d]pyrimidine, and quinoline. This was expected to obtain potent PI3K/mTOR dual inhibitors among them and summarize the structure-activity relationship in order to lay a foundation for further research in the field of treatment of malignant tumors.

Optimization Strategy
Based on the co-crystal structures of the lipid kinase PI3Kα and structures of some known PI3K/mTOR dual inhibitors, it could be found that the structures mainly consisted of three parts (as shown in Figure 2): part A for the affinity binding pocket, part B for the hinge binding pocket, and part C for the ribose binding pocket. Parts A and B were essential to the activity, and the optimization of part C could improve the metabolic stability and oral bioavailability. The available results suggested [14] that when the structure of part A was 2,4-difluoro-N-(2-methoxypyridin-3-yl) benzenesulfonamide, it had the strongest PI3K inhibitory activity. Therefore, the optimization was mainly in parts B and part C.

Optimization Strategy
Based on the co-crystal structures of the lipid kinase PI3Kα and structures of some known PI3K/mTOR dual inhibitors, it could be found that the structures mainly consisted of three parts (as shown in Figure 2): part A for the affinity binding pocket, part B for the hinge binding pocket, and part C for the ribose binding pocket. Parts A and B were essential to the activity, and the optimization of part C could improve the metabolic stability and oral bioavailability. The available results suggested [14] that when the structure of part A was 2,4-difluoro-N-(2-methoxypyridin-3-yl) benzenesulfonamide, it had the strongest PI3K inhibitory activity. Therefore, the optimization was mainly in parts B and part C. In previous studies [15][16][17], the structures of part B were diverse, but they all contained N heteroatoms, which interacted with the key amino acid residue Val851 in the hinge region. In this paper, in order to enhance the binding ability with the hinge region, extended aromatic skeletons and more N heteroatoms were used in the optimization strategy. Unfortunately, the experimental results show that this strategy was not ideal. Compounds with the ever-reported dominant side chain substituents still showed poor enzyme and cell inhibitory activities. It could be concluded that the change of aromatic skeleton would significantly affect the binding ability with receptors and further affected the biological activity of compounds. Therefore, in the subsequent structural optimization, the quinoline skeleton was selected from Omipalisib and used for the following research.
According to reported research [15][16][17], it could be concluded that the introduction of amides and aromatic heterocycles can improve the ligand affinity with receptors. Preliminary calculation research also showed that part C possessed a combining cavity with moderate volume and length, which could accommodate an aromatic heterocycle with an amide. In this paper, the oxazole group with a carboxylic acid ester was creatively introduced as the main fragment at part C. It could effectively occupy the ribose binding pocket and formed a π−π interaction with the amino acids, which may further enhance the affinity with the receptor. In addition, the introduced carboxylic acid ester could be easily converted into various amides as our target compounds. As expected, the hydrophilic group of the introduced amide could not only effectively enhance water H in g e R e g io n In previous studies [15][16][17], the structures of part B were diverse, but they all contained N heteroatoms, which interacted with the key amino acid residue Val851 in the hinge region. In this paper, in order to enhance the binding ability with the hinge region, extended aromatic skeletons and more N heteroatoms were used in the optimization strategy. Unfortunately, the experimental results show that this strategy was not ideal. Compounds with the ever-reported dominant side chain substituents still showed poor enzyme and cell inhibitory activities. It could be concluded that the change of aromatic skeleton would significantly affect the binding ability with receptors and further affected the biological activity of compounds. Therefore, in the subsequent structural optimization, the quinoline skeleton was selected from Omipalisib and used for the following research.
According to reported research [15][16][17], it could be concluded that the introduction of amides and aromatic heterocycles can improve the ligand affinity with receptors. Preliminary calculation research also showed that part C possessed a combining cavity with moderate volume and length, which could accommodate an aromatic heterocycle with an amide. In this paper, the oxazole group with a carboxylic acid ester was creatively introduced as the main fragment at part C. It could effectively occupy the ribose binding pocket and formed a π−π interaction with the amino acids, which may further enhance the affinity with the receptor. In addition, the introduced carboxylic acid ester could be easily converted into various amides as our target compounds. As expected, the hydrophilic group of the introduced amide could not only effectively enhance water solubility and metabolic stability, but also enrich the diversity of the compounds. Herein, a series of new compounds were synthesized.

Structure−Activity Relationship (SAR)
ADP-Glo TM kinase assay and cell viability assay were employed to screen our target compounds. Compared with HS-173 or Omipalisib, the 11a-l and 17a-l compounds showed poor PI3Kα enzyme inhibitory activity and cell proliferation inhibitory activity (as shown in Tables 1 and 2). large volume of substituents, and the small volume of alkyl substituents could not fully fill it either, leading to a significant decline in enzyme inhibitory activity. Compared with HS-173 or Omipalisib, 22c also performed potent enzyme inhibitory activity against mTOR (as shown in Table 4). Most compounds had the same inhibition tendency for the two cell lines with PIK3CA mutation (MCF-7 and HCT-116). Furthermore, most compounds had ClogP values of 4-6, indicating ideal lipid water partition coefficients in order to facilitate cell uptake. large volume of substituents, and the small volume of alkyl substituents could not fully fill it either, leading to a significant decline in enzyme inhibitory activity. Compared with HS-173 or Omipalisib, 22c also performed potent enzyme inhibitory activity against mTOR (as shown in Table 4). Most compounds had the same inhibition tendency for the two cell lines with PIK3CA mutation (MCF-7 and HCT-116). Furthermore, most compounds had ClogP values of 4-6, indicating ideal lipid water partition coefficients in order to facilitate cell uptake. HS-173 or Omipalisib, 22c also performed potent enzyme inhibitory activity against mTOR (as shown in Table 4). Most compounds had the same inhibition tendency for the two cell lines with PIK3CA mutation (MCF-7 and HCT-116). Furthermore, most compounds had ClogP values of 4-6, indicating ideal lipid water partition coefficients in order to facilitate cell uptake.        The negative screening results revealed that the strategies of extension of the aromatic skeleton, as in 11a-l, and the skeleton with more nitrogen atoms, as in 17a-l, should be tentatively used in this study.
For 22a-l, the substituent at the fifth position of oxazole performed crucial effect on the biological activity (as shown in Table 3). The ester showed poor enzyme inhibitory activity while the amides performed enzyme inhibitory activity, which indicated that amide substituents were beneficial to ligand-receptor interaction. Compounds with Nalkyl amides of moderate volume showed ideal inhibitory activity, with isopropyl group as the best, followed by cyclopropyl group. Too small or large alkyl groups would lead to poor inhibitory activity. It was indicated that the ribose binding pocket could not hold a large volume of substituents, and the small volume of alkyl substituents could not fully fill it either, leading to a significant decline in enzyme inhibitory activity. Compared with HS-173 or Omipalisib, 22c also performed potent enzyme inhibitory activity against mTOR (as shown in Table 4). Most compounds had the same inhibition tendency for the two cell lines with PIK3CA mutation (MCF-7 and HCT-116). Furthermore, most compounds had ClogP values of 4-6, indicating ideal lipid water partition coefficients in order to facilitate cell uptake.

Biological Evaluation
In order to further clarify its anti-tumor mechanism, Western blot analysis was used to confirm the anti-tumor mechanism of 22c. As shown in Figure 3, 22c could block the phosphorylation process of AKT at low concentrations, thereby blocking the PI3K/AKT/mTOR signal pathway efficiently. Next, the cell cycle arrest and apoptosis were analyzed by flow cytometry as shown in Figures 4 and 5. 22c could induce apoptosis and inhibited the cell cycle in G0/G1 phase of HCT-116 cells in a dose-dependent manner. Finally, Hoechst33342/PI staining was used to confirm that 22c could significantly affect apoptosis and necrosis of HCT-116 cells (as shown in Figure 6). The above experiments proved that 22c could exert anti-tumor effect by blocking PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR signal pathway efficiently. Next, the cell cycle arrest and apoptosis were analyzed by flow cytometry as shown in Figures 4 and 5. 22c could induce apoptosis and inhibited the cell cycle in G0/G1 phase of HCT-116 cells in a dose-dependent manner. Finally, Hoechst33342/PI staining was used to confirm that 22c could significantly affect apoptosis and necrosis of HCT-116 cells (as shown in Figure 6). The above experiments proved that 22c could exert anti-tumor effect by blocking PI3K/AKT/mTOR signal pathway.

Chemistry
The target compounds could be formed via the Suzuki coupling of the borate of part A and the bromo compound consisting of parts B and C for the sake of the construction of the key carbon-carbon bond. For the synthesis of the borate, 2,4difluorobenzenesulfonyl chloride was condensed with 5-bromo-2-methoxypyridin-3amine (3) [18], and further converted to the borate ester 5 [19] via Miyaura borylation.
Respective routes were employed to synthesize our corresponding three types of target compounds. For the first type, the synthesis of 11a-l was shown in Scheme 1. Compound 8 was prepared via the cyclization of 4-bromo-2-fluorophenylnitrile (6) with ethyl 2-mercaptoacetate and the successive cyclization with formamidine [20], and it was further treated with POCl3 [21] to give chloride 9. 9 was converted to the key intermediates 10a-l via nucleophilic substitution with various nucleophiles, mostly amines. Finally, the target compounds 11a-l were synthesized via Suzuki-Miyaura coupling.

Chemistry
The target compounds could be formed via the Suzuki coupling of the borate of part A and the bromo compound consisting of parts B and C for the sake of the construction of the key carbon-carbon bond. For the synthesis of the borate, 2,4-difluorobenzenesulfonyl chloride was condensed with 5-bromo-2-methoxypyridin-3-amine (3) [18], and further converted to the borate ester 5 [19] via Miyaura borylation.
Respective routes were employed to synthesize our corresponding three types of target compounds. For the first type, the synthesis of 11a-l was shown in Scheme 1.  [20], and it was further treated with POCl 3 [21] to give chloride 9. 9 was converted to the key intermediates 10a-l via nucleophilic substitution with various nucleophiles, mostly amines. Finally, the target compounds 11a-l were synthesized via Suzuki-Miyaura coupling.
Compound 8 was prepared via the cyclization of 4-bromo-2-fluorophenylnitrile (6) with ethyl 2-mercaptoacetate and the successive cyclization with formamidine [20], and it was further treated with POCl3 [21] to give chloride 9. 9 was converted to the key intermediates 10a-l via nucleophilic substitution with various nucleophiles, mostly amines. Finally, the target compounds 11a-l were synthesized via Suzuki-Miyaura coupling.
For the second type, the synthesis of 17a-l was shown in Scheme 2. Arylester 12 was brominated and further cyclized with formamide to obtain 14, which was further converted to 17a-l using the above method.
For the third type, the synthesis of 22a-l was shown in Scheme 3. 18 was deprotonated with LiHMDS and converted to the organic zinc 19 in THF. 19 was brought to the key intermediate 20 by coupling with 6-bromo-4-iodoquinoline via Negishi reaction [22], which was similarly transformed into 22a-l. For the second type, the synthesis of 17a-l was shown in Scheme 2. Arylester 12 was brominated and further cyclized with formamide to obtain 14, which was further converted to 17a-l using the above method.

Molecular Docking
In order to further confirm the ligand-receptor interaction, molecular docking was carried out in AutoDock software. In the binding mode between 22c and PI3Kα (PDB code: 4JPS), the key hydrogen bonds mainly existed in the affinity binding pocket and hinge area binding pocket, which were described as follows (as shown in Figure 7). For the third type, the synthesis of 22a-l was shown in Scheme 3. 18 was deprotonated with LiHMDS and converted to the organic zinc 19 in THF. 19 was brought to the key intermediate 20 by coupling with 6-bromo-4-iodoquinoline via Negishi reaction [22], which was similarly transformed into 22a-l.

Molecular Docking
In order to further confirm the ligand-receptor interaction, molecular docking was carried out in AutoDock software. In the binding mode between 22c and PI3Kα (PDB code: 4JPS), the key hydrogen bonds mainly existed in the affinity binding pocket and hinge area binding pocket, which were described as follows (as shown in Figure 7).

Molecular Docking
In order to further confirm the ligand-receptor interaction, molecular docking was carried out in AutoDock software. In the binding mode between 22c and PI3Kα (PDB code: 4JPS), the key hydrogen bonds mainly existed in the affinity binding pocket and hinge area binding pocket, which were described as follows (as shown in Figure 7).

Molecular Docking
In order to further confirm the ligand-receptor interaction, molecular docking was carried out in AutoDock software. In the binding mode between 22c and PI3Kα (PDB code: 4JPS), the key hydrogen bonds mainly existed in the affinity binding pocket and hinge area binding pocket, which were described as follows (as shown in Figure 7). In the affinity binding pocket, the oxygen atoms of methoxy and sulfonyl formed conservative hydrogen bonds with Lys802; the N atom on the pyridine ring exhibited hydrogen binding with Asp810, Tyr836, and Asp933 through a water molecule. Then, NH of sulfonamide formed a hydrogen bond with Asp933 again. It is very rare to ever report these kinds of multiple hydrogen interactions in the binding pocket.
In the hinge binding pocket, the N atom on the quinoline skeleton formed a conservative hydrogen bond with Val851, which was necessary for maintaining enzymatic potency against PI3Kα. In addition, the benzene ring in the quinoline skeleton also formed a π−π interaction with Tyr836. Although the linker oxazole only formed a weak π−π interaction with Trp780, biological evaluation showed that oxazole linker and amide substituents could make great contributions to the kinase inhibitory activity. It could be inferred that there may be other interactions in the ribose binding pocket. In the affinity binding pocket, the oxygen atoms of methoxy and sulfonyl formed conservative hydrogen bonds with Lys802; the N atom on the pyridine ring exhibited hydrogen binding with Asp810, Tyr836, and Asp933 through a water molecule. Then, NH of sulfonamide formed a hydrogen bond with Asp933 again. It is very rare to ever report these kinds of multiple hydrogen interactions in the binding pocket.
In the hinge binding pocket, the N atom on the quinoline skeleton formed a conservative hydrogen bond with Val851, which was necessary for maintaining enzymatic potency against PI3Kα. In addition, the benzene ring in the quinoline skeleton also formed a π−π interaction with Tyr836. Although the linker oxazole only formed a weak π−π interaction with Trp780, biological evaluation showed that oxazole linker and amide substituents could make great contributions to the kinase inhibitory activity. It could be inferred that there may be other interactions in the ribose binding pocket.
In the binding mode between 22c and mTOR (PDB code: 4JT6), the N atom on the quinoline core formed a critical hydrogen bond with Val2240. The binding model proposed above could support the effect of 22c on PI3Kα and mTOR kinase.

Materials and Methods
The reagents and solvents used in this article were chemically pure or analytically pure, and could be used directly without purification. The silica gel used for flash column chromatography and thin layer chromatography was purchased from Qingdao Yumingyuan Silicone Chemical Factory (Qingdao, China). The NMR spectra was recorded by a Bruker AV-400/600 nuclear magnetic resonance instrument, in which TMS was used as the internal standard, the chemical shift is expressed in ppm(δ), and the coupling constant (J) is expressed in Hertz (Hz). The HRMS spectrum was determined by an Agilent Accurate Mass Q-TOF 6530 mass spectrometer (Agilent, Santa Clara, CA, USA).
The synthesis of 6-bromo-N-isopropylpyrido[2,3-d]pyrimidin-4-amine (16a). Isopropyl amine (86 µL, 1 mmol) was added to a mixture of 15 (75 mg, 0.25 mmol) and Et 3 N (70 µL, 0.5 mmol) in THF (5 mL) under ice cooling bath. Then, the mixture was gradually raised to room temperature, and stirred for an additional 4 h. It was concentrated in vacuo and the residue was stirred in ethyl acetate (5 mL) and H 2 O (5 mL). The organic layer was separated and dried. The solvent was evaporated in vacuum to afford 16a as an ashy solid (55 mg, 83.1%), which was used without further purification.
16b−l were prepared by a similar procedure described for the synthesis of 16a.  General Procedure C was used for the synthesis of 22a-l. The synthesis of ethyl 2-(6-bromoquinolin-4-yl)oxazole-5-carboxylate (20). To a mixture of zinc chloride (1.0 M in THF, 6 mL,6 mmol) and compound 18 (242 µL, 2 mmol) in THF (10 mL) at −10 • C under argon, lithium hexamethyldisilazide (1.0 M in THF, 3 mL, 3 mmol) was added and the mixture was stirred at −10 • C for 1 h. Then, 6-bromo-4iodoquinoline (333 mg, 1 mmol) and Pd(PPh 3 ) 4 (116 mg, 0.1 mmol) were added and the mixture was stirred at 60 • C for 15 h. It was cooled to room temperature and neutralized by saturated ammonium chloride (30 mL). Ethyl acetate (10 mL) was added and the mixture was stirred for 15 min. The separated organic layer was evaporated in vacuum and the residue was purified by flash column chromatography to obtain a white solid (20) (245mg, 71%). 1  The synthesis of 2-(6-bromoquinolin-4-yl)-N-isopropyloxazole-5-carboxamide (21c). In a sealed tube, a mixture of ester 20 (88 mg, 0.25 mmol) and isopropyl amine (1 mL) was stirred at 100 • C for 4 h. It was cooled to room temperature and H 2 O (5 mL) was added. The suspension was filtered and dried to give an ashy solid 21c (76.3 mg, 86%), which was used without purification.
21a-b, 21d-l were prepared by a similar procedure described for the synthesis of 21c.